Liver X receptor inhibition potentiates mitotane-induced adrenotoxicity in ACC

Abstract

Adrenocortical carcinoma (ACC) is a rare aggressive malignancy with a poor outcome largely due to limited treatment options. Here, we propose a novel therapeutic approach through modulating intracellular free cholesterol via the liver X receptor alpha (LXRα) in combination with current first-line pharmacotherapy, mitotane. H295R and MUC-1 ACC cell lines were pretreated with LXRα inhibitors in combination with mitotane. In H295R, mitotane (20, 40 and 50 µM) induced dose-dependent cell death; however, in MUC-1, this only occurred at a supratherapeutic concentration (200 µM). LXRα inhibition potentiated mitotane-induced cytotoxicity in both cell lines. This was confirmed through use of the CompuSyn model which showed moderate pharmacological synergism and was indicative of apoptotic cell death via an increase in annexinV and cleaved-caspase 3 expression. Inhibition of LXRα was confirmed through downregulation of cholesterol efflux pumps ABCA1 and ABCG1; however, combination treatment with mitotane attenuated this effect. Intracellular free-cholesterol levels were associated with increased cytotoxicity in H295R (r2 = 0.5210) and MUC-1 (r2 = 0.9299) cells. While both cell lines exhibited similar levels of free cholesterol at baseline, H295R were cholesterol ester rich, whereas MUC-1 were cholesterol ester poor. We highlight the importance of LXRα mediated cholesterol metabolism in the management of ACC, drawing attention to its role in the therapeutics of mitotane sensitive tumours. We also demonstrate significant differences in cholesterol storage between mitotane sensitive and resistant disease.

Novel Insights into the Molecular Regulation of Ribonucleotide Reductase in Adrenocortical Carcinoma Treatment

“…However, in this context, it is relevant to mention, that the preclinical screening platform in the field of ACC was inadequate for a long time, as we faced a huge lack of tumor models, with NCI-H295R as the only available cell line of human origin for more than 20 years [17]. In the current study, we have performed an extensive preclinical drug screening and subsequent mechanistical investigation based on classical chemotherapies and innovative targeted agents, using two different adrenocortical cancer cell lines: the classical NCI-H295R cell line and the more recently developed and highly drug-resistant MUC-1 cell line [11,[18][19][20][21][22][23].…”

“…Cells with defective levels of ATR signaling commonly show a highly increased sensitivity to replication stress and many DNA-damaging agents and antimetabolites, such as gemcitabine [31,32]. This could explain, furthermore, the frequently observed higher drug sensitivity of NCI-H295 compared to MUC-1 [11,[18][19][20][21][22][23]. However, as there is also a strong interplay between ATR and the p53 status [33], and as both cell lines carry, furthermore, different p53 mutations, this needs to be investigated in more detail in further studies.…”

Molecular Mechanisms of Mitotane Action in Adrenocortical Cancer Based on In Vitro Studies

“…Curiously, despite the strong induction of lipid peroxidation, mitotane does not induce ferroptosis [ 64 , 65 ]. Since mitotane increases free cholesterol in cells and oxysterols, such as 27-hydroxycholesterol, which could reduce this process [ 66 ], the cholesterol metabolism could be an interesting druggable pathway to counteract mitotane resistance in ACC. On these bases, the introduction of LXRα and PCSK9 inhibitors as future therapeutic approaches could be a promising tool to reduce mitotane resistance and/or to optimize its therapeutic dose [ 46 , 66 ].…”

“…Since mitotane increases free cholesterol in cells and oxysterols, such as 27-hydroxycholesterol, which could reduce this process [ 66 ], the cholesterol metabolism could be an interesting druggable pathway to counteract mitotane resistance in ACC. On these bases, the introduction of LXRα and PCSK9 inhibitors as future therapeutic approaches could be a promising tool to reduce mitotane resistance and/or to optimize its therapeutic dose [ 46 , 66 ]. In the adrenal gland, the role of LXRα and its oxysterol ligands are critically important in the fine regulation of cholesterol efflux since the excess free cholesterol in cells is converted into oxysterols through the action of enzymes, such as CYP27A1.…”

“…Pharmacological inhibition of LXRα significantly reduces the expression of the cholesterol efflux pump (ABCA1 and ABCG1) and is accompanied by higher intracellular free cholesterol concentrations, ER stress, apoptosis, and cell death markers expression. This effect is complementary to mitotane-induced lipotoxicity, and, using a combined therapeutic approach, lower doses of mitotane can be expected to be used, resulting in reduced toxicity [ 66 ].…”